Cyclic AMP and the Control of the Meiotic Cell Cycle

描述(由申请人提供)：本提案侧重于雌配子细胞减数分裂周期中G2/M转变的调节机制。这是一个发生在体细胞创造的明确定义的“生态位”中的过程，可以用作细胞与细胞相互作用的范例。了解其中所涉及的信号将为cAMP如何在多细胞结构中调节细胞周期的进入和退出提供一个模型，从而揭示调节细胞复制的旁分泌机制。在过去的五年里，我们已经确定了控制小鼠卵母细胞MPF(细胞周期蛋白B/cdc2复合体)活性的cAMP信号级联中的关键成分。我们的遗传模型表明，这些成分在体内被灭活，卵母细胞中cAMP信号的中断与女性不孕症的减少或完全有关，并阻止了G2/M转变。我们建议使用这些模型以及我们产生的生化工具来了解cAMP如何作为重新进入减数分裂细胞周期的信号，以及体细胞如何在生殖细胞中控制这一途径。实验计划被组织成三个具体目标。第一个具体目标将致力于了解卵母细胞中cAMP合成是如何调节的。GPCRs缺陷小鼠以及在卵母细胞中表达的PDE将被分析以恢复减数分裂，以及与细胞内cAMP水平和细胞周期调节因子活性有关的细胞周期状态。此外，还将研究体细胞产生的GPCRs及其配体的性质。第二个具体目标将致力于了解PDE3A是如何通过磷酸化来调节的。PDE3A是小鼠卵母细胞中主要的PDE，是减数分裂成熟所必需的。最后一个具体目标将侧重于库尔德工人党下游营地梯级的步骤。将分析PKA对双磷酸酶cdc25B和新发现的激酶Wee1B的磷酸化的影响及其在卵母细胞中的定位。还将探讨这两种酶的活性和定位对MPF激活状态的影响。所提出的研究将进一步加深我们对特化减数分裂细胞周期的调节以及

DESCRIPTION (provided by applicant): The present proposal focuses on the mechanisms of regulation of the G2/M transition in the specialized meiotic cell cycle of the female gamete. This is a process that occurs in a well defined "niche" created by somatic cells that can be used as a paradigm of cell-cell interaction. Understanding the signaling involved will provide a model for how cAMP regulates entry and exit from the cell cycle in a multicellular structure thereby shedding light on paracrine mechanisms of regulation of cell replication. Over the past five years, we have identified key components in the cAMP signaling cascade that control the activity of MPF (cyclinB/cdc2 complex) in mouse oocytes. Our genetic models where these components have been inactivated in vivo show that disruption in cAMP signaling in the oocytes is associated with reduced or complete female infertility and blockade in G2/M transition. We propose to use these models as well as biochemical tools that we have generated to understand how cAMP functions as a signal for reentry into the meiotic cell cycle and how somatic cells control this pathway in germ cells. The experimental plan is organized into three Specific Aims. The first Specific Aim will be devoted to understand how cAMP synthesis is regulated in oocytes. Mice defective in GPCRs as well PDEs expressed in oocytes will be analyzed for meiotic resumption and the state of the cell cycle related to intracellular cAMP levels and activity of cell cycle regulators. In addition, the properties of the GPCRs and their ligands produced by somatic cells will be investigated. The second Specific Aim will be devoted to understanding how PDE3A is regulated by phosphorylation. PDE3A is the major PDE identified in mouse oocytes, which is indispensable for meiotic maturation. The last Specific Aim will focus on the steps in the cAMP cascade downstream of PKA. The effect of PKA phosphorylation of the dual phosphatase cdc25B and the newly discovered kinase Wee1 B as well as their localization in the oocytes will be analyzed. The impact of the activity and localization of these two enzymes on the MPF state of activation also will be explored. The studies proposed will further our understanding of the regulation of the specialized meiotic cell cycle as well as the